Functional Rescue of Mutant V2 Vasopressin Receptors Causing Nephrogenic Diabetes Insipidus by a Co-Expressed Receptor Polypeptide
| Title: | Functional Rescue of Mutant V2 Vasopressin Receptors Causing Nephrogenic Diabetes Insipidus by a Co-Expressed Receptor Polypeptide |
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| Authors: | Schoneberg, Torsten; Yun, June; Wenkert, M.D., Ph.D., David; Wess, Jurgen |
| Publisher: | Embo Journal |
| Date Published: | March 15, 1996 |
| Reference Number: | 28 |
Inactivating mutations in distinct G protein-coupled receptors (GPCRs) are currently being identified as the cause of a steadily growing number of human diseases. Based on previous studies showing that GPCRs are assembled from multiple independently stable folding units, we speculated that such mutant receptors might be functionally rescued by 'supplying' individual folding domains that are lacking or misfolded in the mutant receptors, by using a co-expression strategy. To test the feasibility of this approach, a series of nine mutant V2 vasopressin receptors known to be responsible for X-linked nephrogenic diabetes insipidus were used as model systems. These mutant receptors contained nonsense, frameshift, deletion or missense mutations in the third intracellular loop or the last two transmembrane helices. Studies with transfected COS-7 cells showed that none of these mutant receptors, in contrast to the wild-type V2 receptor, was able to bind detectable amounts of the radioligand, [3H] arginine vasopressin, or to activate the G(S)/adeylyl cyclase system. Moreover, immunological studies demonstrated that the mutant receptors were not trafficked properly to the cell surface. However, several of the nine mutant receptors regained considerable functional activity upon co-expression with a C-terminal V2 receptor peptide spanning the sequence where the various mutations occur. In many cases, the restoration of receptor activity by the co-expressed receptor peptide was accompanied by a significant increase in cell surface receptor density. These findings may lead to the design of novel strategies in the treatment of diseases caused by inactivating mutations in distinct GPCRs.
This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
Mutations in G protein-coupled receptors (GPCRs) (the family of which V2 receptor is a member) are currently being identified as the cause of a growing number of human diseases. GPCRs are located in specific cell membranes, the thin band that encircles a cell separating the cell from its environment. If you think of the GPCR as a string (it is actually a chain of polypeptides), the majority of the GPCR lies within the cell membrane in seven distinctly folded clumps called transmembrane helices. Part of the GPCR snakes outside the cell forming three curves called extracellular loops 1, 2, and 3. Part of it snakes inside the cell forming three curves called intracellular loops 1, 2, and 3. One end of the GPCR, called the amino terminus, is outside the cell with the extracellular loops; the other end, called the carboxy terminus, is inside the cell with the intracellular loops. (Please see diagram of GPCR.)
In most cases, mutations in the GPCRs result in shortened or misfolded receptor proteins which are unable to bind smaller molecules and/or couple with G proteins. Schoneberg, et al., tested to see if mutated, therefore nonfunctional, GPCR receptors could be restored to function properly by supplying them with the correctly folded transmembrane domain (which is missing or misfolded in the mutant receptors).
They tested their hypothesis on mutated V2 receptor genes known to be the cause of nephrogenic diabetes insipidus (NDI), a rare kidney disease characterized by the V2 receptor being unable to bind with vasopressin and resulting in the kidneys' inability to concentrate urine. The nine different types of mutated V2 receptors in this study all contained mutations in the same area -- either the third intracellular loop or the last two transmembrane domains. None of the mutant receptors were able to bind to VP or to couple with their respective G protein. All the mutant V2 receptors were found at high levels in the cell interior, but they were found at much lower levels at the cell surface, indicating that the mutant receptors could not get to the cell surface. This suggests that the endoplasmic reticulum inside the cell can recognize, retain, and possibly degrade improperly folded receptor proteins. However, the lack of receptors at the cell surface is not sufficient to account for the lack of functional activity found with all nine mutant receptors.
The authors knew that all the mutant receptor genes had these mutations in the same area and reasoned that if they blended the mutants with fragments of healthy V2 receptors that contained the genetic sequences missing in the mutant V2 receptors, then the mutated receptors, being expressed with the parts they were lacking, would be able to function normally. This strategy worked for four of the mutant V2 receptors, enabling them to have a high level of binding sites, an increased ability to couple with their G protein, and an increased ability to move to the cell surface. Their findings suggest that the ability of the healthy V2 fragment to restore function to mutant V2 receptors containing mutations in their third intracellular loop or last two transmembrane domains is due to the formation of specific complexes between the fragment and portions of the mutant V2 receptor. In short, the fragment and the mutant receptor blend and reconstitute themselves to be a more functional whole.
Even the five mutant receptors that did not respond as well to the healthy V2 fragment gained in their ability to interact with G proteins. That this sort of functional rescue is possible holds great possibilities for therapy as many diseases are now known to result from mutated, therefore nonfunctional, GPCRs.